ImageJ: Wrong transferrence of Coordinates from picture to picture - macros

Question

for my Thesis I must count cells on pictures stained with Immunofluorescence and I am writing a macro in ImageJ to do it for me.
For this I coloursplit the picture, analyse particles in the red channel (my Antibody´s colour) and then I want to take the coordinates of analysed particles and only count them if at the same coordinates in the blue channel there is also a staining (DAPI - just a general cell staining).
This way I assure that there is as little dirt counted as possible.
The problem is that when I get the coordinates from the results table and use them to makePoint(x,y) the coordinates are "distorted" - usually the correct coordinates but plus 4ish, though never in exactly the same, which is why i can´t simply distract a number from the coordinates
Below I first write down the critical lines of code, then the whole code.
Thank you very much in advance
run("Analyze Particles...", " circularity=r1-r2 display clear in_situ");
roiManager("deselect");
z=nResults;
for (j=0; j<z; j++) { //loops through the Results table and adds to "counter" if a match is found
selectImage(channelsplit[2]); //selects blue window
setThreshold(d,l);
run("Threshold...");
setOption("BlackBackground", true);
run("Convert to Mask", "method=Default background=Default black");
run("Coordinates...", "left=0 right=19373 top=0 bottom=13600"); //I tried to set the boundaries of the pictures equally, but it didnt work
makePoint(getResult("X", j)), (getResult("Y", j));
print ("X" + j + ": " + getResult("X",j)); //this and the following line are not to be in the program, once it works
print ("Y" + j + ": " + getResult("Y",j));
run ("Measure");
if (getResult("Mean", nResults-1)>100) {
counter++;
}
} //for j
print (i + ": " + counter);
setBatchMode(true);
if (isOpen("ROI Manager")) {
r=roiManager("count");
} else {
setBatchMode(false);
exit("You need a ROI Manager open with the ROIs of all the pictures to be measured");
}
Dialog.create ("Variables")
Dialog.addCheckbox ("red", true);
Dialog.addCheckbox ("green", false);
Dialog.addCheckbox ("blue", false);
Dialog.addCheckbox ("watershed", true);
Dialog.addNumber ("Thresholddark:", 110);
Dialog.addNumber ("Thresholdlight:", 255);
Dialog.addNumber ("greenThresholddark:", 110);
Dialog.addNumber ("greenThresholdlight:", 253);
Dialog.addNumber ("pmin:", 15);
Dialog.addNumber ("pmax:", 100);
Dialog.addNumber ("roundness1:", 0.5);
Dialog.addNumber ("roundness2:", 1.0);
Dialog.addMessage("batch - select 'true' for your macro to run faster, but you will not see what it does until finished\nstack - selecht this box if you use unstacked pictures and want them stacked, deselect if you use a readied stack or a single picture\nred - select this box if you want to count red coloured cells \ngreen - select this box if you want to count green coloured cells \nblue - select this box if you want to count blue coloured cells\nall variables are the same for the colours you count. It may be better to adjust variables for each colour and count seperately\nwatershed - select this box only if you have overlapping cells in your image\nThresholdlight - particles lighter than this won't be measured/counted \nThresholddark - particles darker than this won't be measured/counted \nThresholdlight and Thresholddark can be between 0 and 255 \ntry thresholding manually at least once manually to get best results \npmin - particles with a size smaller than this won't be counted \npmax - particles with a size greater than this won't be counted")
Dialog.show ();
cr=Dialog.getCheckbox();
cg=Dialog.getCheckbox();
cb=Dialog.getCheckbox();
w=Dialog.getCheckbox();
d=Dialog.getNumber();
l=Dialog.getNumber();
dg=Dialog.getNumber();
lg=Dialog.getNumber();
pmin=Dialog.getNumber();
pmax=Dialog.getNumber();
r1=Dialog.getNumber();
r2=Dialog.getNumber();
dir = getDirectory("Choose a Directory "); //choose the folder with all the pictures to be measured
listFiles(dir); //I found this in a listFiles recursively Demo and changed it to open my pictures
function listFiles(dir) {
list = getFileList(dir);
for (o=0; o<list.length; o++) { //loops through the file list, opening then analysing then closing one after another
if (endsWith(list[o], "/")) {
listFiles(""+dir+list[i]);
} else {
open(dir + list[o]);
//the previous opens all pictures in a given folder in a way i do not understand
//I only use this function because I could not use the variable list.length outside of it for unknown reasons
//following is my cellcount program to be executed for each picture
//then the picture will be closed before the new one is opened
run("Split Channels");
channelsplit = getList("image.titles");
if (cr==1) {
a=0;
}
if (cg==1) {
a=1;
}
selectImage(channelsplit[a]); //selects red or green window, depending on input in the Dialog
setThreshold(d,l);
run("Threshold...");
setOption("BlackBackground", true);
run("Convert to Mask", "method=Default background=Default black");
run("Coordinates...", "left=0 right=19373 top=0 bottom=13600");
for (i=2*o; i<=((2*o)+1); i++) { //loops through two ROIs of the ROIManager
counter=0;
selectImage(channelsplit[a]);
roiManager("deselect");
roiManager("select", i);
if (w==1) {
run("Watershed", "slice");
}
run("Analyze Particles...", " circularity=r1-r2 display clear in_situ");
roiManager("deselect");
z=nResults;
for (j=0; j<z; j++) { //loops through the Results table and adds to "counter" if a match is found
selectImage(channelsplit[2]); //selects blue window
setThreshold(d,l);
run("Threshold...");
setOption("BlackBackground", true);
run("Convert to Mask", "method=Default background=Default black");
run("Coordinates...", "left=0 right=19373 top=0 bottom=13600"); //I tried to set the boundaries of the pictures equally, but it didnt work
makePoint(getResult("X", j)), (getResult("Y", j));
print ("X" + j + ": " + getResult("X",j)); //this and the following line are not to be in the program, once it works
print ("Y" + j + ": " + getResult("Y",j));
run ("Measure");
if (getResult("Mean", nResults-1)>100) {
counter++;
}
} //for j
print (i + ": " + counter);
close("Results"); //closes Results to get the variables in order for the next window
// IJ.renameResults("res" + i);
} //for i
close("*"); //closes all image windows to save RAM
} //else
} //for o
} //function
setBatchMode(false);